ABSTRACT
Objective To construct a eukaryotic expression plasmid of pcDNA 3.1-Ag85A-CD226, and to use it as DNA vaccine then further evaluate its immunogenicity through oral administration in a mouse model.Methods The CD226-PCR2.1-ToPo plasmid was used as the template to clone CD 226 gene by PCR.The CD226 gene was then inserted into pcDNA 3.1-Ag85A plasmid to construct the recombinant plas-mid of pcDNA3.1-Ag85A-CD226.After identified by restriction enzyme analysis and sequencing , the re-combinant plasmid was transfected into HEK 293 cells by using lipofection .The expression of Ag85A-CD226 gene in HEK293 cells was detected by RT-PCR, Western blot and indirect immunofluorescence assay .The purified recombinant plasmid was used to prepare the Ag 85A-CD226 DNA vaccine by liposomal encapsula-tion.The vaccine was administered intragastrically to mice .The activities of NK cells , the cytokine levels in the supernatants of spleen cell cultures and the mRNA level of cytokines in the intestines were evaluated to analyze the immunogenicity of Ag85A-CD226 DNA vaccine.Results The Ag85A-CD226 DNA vaccine was prepared successfully .The expression of Ag85A-CD226 fusion protein was detected in HEK293 cells.The activities of NK cells from mice vaccinated with Ag 85A-CD226 DNA vaccine were higher than those from other control groups (P0.05).Conclusion The Ag85A-CD226 DNA vaccine could significantly enhance Th1 type immune responses systemically and in the intestine as in comparison with those vaccinated with single dose of Ag 85A DNA vaccine or CD226 DNA vaccine.
ABSTRACT
The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Antigens, Bacterial , Mycobacterium tuberculosis/immunology , Peptides , Tuberculosis/diagnosis , Antigens, Bacterial/immunology , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Peptides/immunology , Sensitivity and Specificity , Tuberculosis, Pulmonary/diagnosis , Tuberculosis, Pulmonary/immunology , Tuberculosis/immunologyABSTRACT
Objective To obtain M. Tuberculosis Ag85A protein by prokaryotic expression. Methods The fbpA gene encoding M. Tuberculosis Ag85A protein was amplified by polymerase chain reaction ( PCR) from M. Tuberculosis H37R_V strain. The PCR product was cloned into prokaryotic expression vector pProEXHTb to generate the recombi-nant plasmid pProfbpA, which was then transformed into the competence Escherichia coli BL21 cells. The recombi-nant Ag85A protein was successfully expressed by isopropyl thio-β-D-galactoside(IPTG) induction and purified by the Ni-purification system. The distribution of fbpA gene in different nonpathogenic mycobacterial strains was screened by PCR and ELISA was performed to determine the immunoreactivity of the recombinant Ag85A protein with serum from mice with mycobacterial infections. Results 32 ku Ag85A protein was successfully expressed and purified. It was confirmed by PCR and ELISA that fbpA gene presented in the genomes of M. Tuberculosis H37Rv, H37Ra, BCG, M. Smegmatis, M. Terra, M. Trivial and M. Phlei, but being absent in the genomes of M. Vaccae. The highest Ag85 A antibody titer was found in serum of TB patients and mice infected by M . Tuberculosis H37Rv .Conclusion The recombinant Ag85A protein was successfully expressed and purified.
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Objective: To clone the Ag85 A antigen gene of Mycobacterium tuberculosis and express it in E. coli k802. Methods: The Ag85 A gene was amplified from the genome of Mycobacterium tuberculosis by PCR and was inserted into prokaryotic expressing vector pGEX5T to construct pGEX5T-Ag85A recombinant plasmid. The expression of Ag85A protein in E. coli k802 was induced by IPTG. The expressed protein was purified by affinity chromatography and the antigenicity of the expressed protein was confirmed by Western blotting assay. Results: A band about 0.9 kb in length was obtained by PCR and the recombinant plasmid pGEX5T-Ag85A was successfully constructed. A new band about 58 000 in length was observed after IPTG induction in E. coli. The relative molecular weight of the expressed protein was consistent with that expected. A single protein band of 58 000 in length was obtained after purification. The expressed protein could be specifically recognized by the sera of patients with tuberculosis patients. Conclusion: The Ag85A gene of Mycobacterium tuberculosis has been successfully cloned and expressed in E. coli k802, which paves a way for further studies on diagnosis and therapy of tuberculosis.
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Objective: To use in vivo fluorescence image analysis system for evaluating the efficacies of pVAX1-Ag85A and pVAX1-Ag85B DNA vaccines in treatment of bladder cancer cell-implanted tumors in mice. Methods: Discosomasp red fluorescent protein (DsRed) stably transfected bladder cancer BTT cell line (BTT-DsRed) was established and BTT-DsRed cell-implanted mouse model was constructed. Six days later, 24 BTT-DsRed-bearing mice were randomly divided into pVAX1-Ag85A DNA vaccine group, pVAX1-Ag85B DNA vaccine group, and saline group through injecting the pVAX1-Ag85A, pVAX1-Ag85B, and saline into the right hind limbs of mice, respectively. The growth and metastasis of implanted BTT-DsRed tumors were examined by in vivo fluorescence image analysis system. Results: BTT cell line stably transfected with DsRed (BTT-DsRed) was successfully established. Fluorescence visible mouse model was successfully es-tablished by inoculating BTT-DsRed cells into hind limbs of mice. After treatment with pVAX1-Ag85A or pVAX1-Ag85B for 2 weeks, the in vivo tumor fluorescence intensity in pVAX1-Ag85B group was significantly lower than that in the saline group (P <0.05). After 3 weeks, tumor fluorescence intensities in both pVAX1-Ag85A and pVAX1-Ag85B groups were significantly lower than that in the saline group (P < 0.01). But the efficacies of pVAX1-Ag85A and pVAX1-Ag85B groups were similar (P > 0.05). The distant lymphatic metastasis rate in pVAX1-Ag85B group was significantly lower than those in the saline (25.0% vs 87.5%) and pVAX1-Ag85A groups (25.0% vs 62.5%) (P <0.05). Conclusion: In vivo fluorescence image analysis system can dynamically, sensitively and visually evaluate the anti-tumor effects of DNA vaccines against bladder cancer cell-implanted tumors. Both pVAX1-Ag85A and pVAX1-Ag85B DNA vaccines have anti-tumor effects for bladder cancers.